The ES proteins were precipitated using trichloroacetic acid (TCA) and acetone using a previously described method with some modifications [27 (link)]. The electrophoresis was performed as described previously [17 (link),28 (link)]. Briefly, 300 or 200 μg of ES proteins were loaded onto 11-cm pH 4–7 immobilized pH gradient (IPG) strips (Bio-Rad, USA) and separated by isoelectric focusing (IEF). IEF was performed using a Protean IEF Cell at 20°C as follows: S1: 50 V, 12 h; S2:250 V, 30 min; S3: 1 000 V, 30 min; S4: 8 000 V, 4 h; and S5: 8 000 V, 40 000 Vh (using a limit of 50 μA/strip). SDS-PAGE was performed with 10% gels using a Mini Protean cell (Bio-Rad, USA). Three replicates were run for the sample. After 2D gel electrophoresis, proteins were either stained with Coomassie blue R-250 for proteomic analysis or used for immunoblotting as previously described [29 (link)]. Both the 2-DE and immunoblotting tests were repeated three times, with no variation in results observed. Images of immunoblots were captured using ImageScanner (GE healthcare, USA) and aligned with equivalent protein stained 2-DE gels using Image Master 2D Platinum 6.0 (GE healthcare, USA).
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